The technologies presented in this application have the potential to greatly expand the molecular toolbox available to the researchers and pathologists who analyze cancer patient tissue specimens. Our proposal details the development of a new methodology to simultaneously measure the expression of a large number of genes in formalin fixed and paraffin embedded (FFPE) tissue sections. Alternative approaches begin with extraction of damaged, relatively low quality RNA, which limits downstream analysis. In contrast, our system takes advantage of the crosslinked RNA molecules to obtain amplifiable signals in situ, which provides several distinct advantages related to assay sensitivity and workflow simplicity. Importantly, our system utilizes widely available instrumentation and reagents, making it immediately accessible to researchers and clinicians. We seek to achieve three overlapping goals in this project: 1. Development of a robust system to generate PCR amplifiable signals in situ using FFPE tumor sections. The success of this effort will provide the research and clinical community with a completely new method of performing gene expression analysis of FFPE specimens and fixed suspension cells. 2. Integration with high throughput DNA sequencing analysis. High throughput, or next generation DNA sequencing (NGS) is a powerful technology for analyzing complex mixtures of DNA sequences. We propose to integrate our new methodology with NGS analysis for the highly multiplexed measurement of gene expression in FFPE specimens. 3. Multiplexed measurement of spatially resolved gene expression. This part of the project is devoted to the development of a novel methodology for measuring a large number of genes in FFPE sections or fixed cell spreads, while retaining single cell resolution of the original tissue. To this end, e propose to adapt recently developed techniques for 'local' DNA amplification. Such a system has the potential to dramatically increase the amount of information that researchers and pathologists are able to glean by analyzing a single microscope slide.